The objective of this proposal is to develop new probe targeting methodology based on the enzyme lipoic acid ligase (LplA). LplA catalyzes the site-specific covalent conjugation of the cofactor lipoic acid onto the lysine sidechain of one of three bacterial protein substrates. We propose to re-engineer LplA for probe targeting inside living mammalian cells. We previously engineered a transposable 12-amino acid peptide substrate for LplA, and mutagenized the lipoate binding pocket to accommodate a coumarin fluorophore. Here, we propose to (1) use a combination of rational design and directed evolution to engineer LplA-derived ligases for the red fluorophore resorufin and the photocrosslinker benzophenone. For directed evolution, we will explore FACS, cell laser ablation, and antibiotic selection strategies in parallel; (2) develop two-step probe targeting schemes based on LplA-catalyzed ligation of functional group handles such as alkyl azides; and (3) apply our new probe ligases to the study of neurexin and neuroligin biology in living neurons. Our work should benefit cell biologists interested in minimally- invasive analysis of protein function in single living cells, and neuroscientists interested in understanding the molecular bases of synapse development. PUBLIC HEALTH RELEVANCE: We are proposing new chemical technology that has the potential to transform biological microscopy. As an alternative to Green Fluorescent Protein, which received this year's Nobel Prize in Chemistry, our probe ligase technology is far less invasive to proteins under study, and can introduce a much wider variety of fluorophore, photocrosslinker, and other probe structures with superior properties to the GFP chromophore. In this proposal, we also plan in-depth studies of neurexin and neuroligin biology, using probe ligase technology; these studies will shed light on the molecular mechanisms of synapse development and could accelerate the discovery of drugs to treat diseases such as autism.